The present invention relates to a method for specifically cleaving a double-stranded DNA and a kit for the cleaving method.
Specific cleavage of double-stranded DNA is an indispensable means for gene analysis in recombinant DNA experiments and in genome projects for living organisms including the Human Genome Project. Potent tools for such a specific cleavage of DNA include class II restriction enzymes cleaving double-stranded DNAs at their specific positions. Known enzymes cleave more than about 60 different specific nucleotide sequences of DNA. Indeed, restriction enzymes cleaving DNAs at specific positions can be advantageously used at certain aspects of the above-described recombinant DNA experiments and genome projects, but these enzymes cannot cleave DNAs having no specific recognition sites, and, on the contrary, cannot cleave only a single recognition site of DNAs having plural same recognition sites.
In order to more efficiently use the gene information obtained from the genome project as described above, for example, it is necessary to establish techniques for specifically cleaving double-stranded DNAs at the desired positions. As such techniques, a chemical method mediated by forming a three-stranded DNA (S. A. Strobel et al., Science, 249, 73-75 (1990)) and an enzymatic method similarly mediated by forming a three-stranded DNA have been proposed. Interesting examples of the latter enzymatic method include that comprising protecting the site to be cleaved of a double-stranded DNA by forming the three-stranded DNA using the recA protein involved in the homologous recombination and a single-stranded DNA having a homologous sequence to the double-stranded DNA, treating the double-stranded DNA excluding the three-stranded portion with methylase, allowing the three-stranded portion to dissociate, and cleaving only this portion at a single site with a restriction enzyme (L. J. Ferrin, et al., Science, 254, 1494-1497 (1991)). Another known method is a method for forming a three-stranded DNA of Hoogsteen or reverse Hoogsteen base paired structure between a double-stranded DNA having poiypurine/polypyrimidine sequence and a single-stranded DNA of polypyrimidine.
The chemical method is excellent for specifically cleaving DNA, but its cleavage efficiency is generally low. On the other hand, the above-described enzymatic method is excellent in cleaving DNA at only a single site. This method is, however, disadvantageous in that it requires the methylase treatment for the site-specific cleavage of DNA with a restriction enzyme and that it is limitedly applicable to DNA having a specific site cleavable with such a restriction enzyme.
Development of techniques to specifically, simply, and efficiently cleave DNA at the desired position regardless of nucleotide sequence has thus been desired.
The present inventor has found that a double-stranded DNA having a three-stranded DNA portion comprising the double-stranded DNA and a single-stranded DNA or PNA can be specifically cleaved at the phosphodiester bond at any position of or adjacent to the three-stranded DNA portion directly with a certain nuclease without the methylase treatment. The present inventor has also discovered that the resistance of double-stranded DNA or double-stranded DNA portion to the cleavage with a nuclease is enhanced in the presence of a homologous recombination protein, which can stimulate the three-stranded DNA formation, and nucleoside triphosphate or its analogue. This invention has been completed based on such findings.
This invention relates to a method for specifically cleaving a double-stranded DNA using an enzyme, the method comprising the steps of:
(A) forming a complex having a three-stranded DNA portion comprising a double-stranded DNA to be cleaved and a single-stranded DNA molecule containing a nucleotide sequence substantially homologous to a specific region of the nucleotide sequence of the double-stranded DNA or a PNA containing a nucleotide sequence substantially homologous to a specific region of the nucleotide sequence of the double-stranded DNA;
(B) cleaving the complex thus obtained with a nuclease capable of recognizing the three-stranded DNA portion in the double-stranded DNA and of cleaving any one of the phosphodiester bonds in or adjacent to the three-stranded DNA portion; and
(C) optionally inactivating the nuclease.
In a preferred embodiment of this invention, the above-described step of forming a complex having a three-stranded DNA portion comprising a double-stranded DNA and single-stranded DNA molecule is performed in the presence of a homologous recombination protein and nucleoside triphosphate or its analogue.
In another aspect, this invention relates to a method for enhancing the resistance of a double-stranded DNA against the cleavage with a nuclease by adding a homologous recombination protein and nucleoside triphosphate in a composition containing a double-stranded DNA.
In yet another aspect, this invention relates to a kit for specifically cleaving a double-stranded DNA using an enzyme, comprising:
(a) a nuclease capable of recognizing a three-stranded DNA portion of a double-stranded DNA in a complex having the three-stranded DNA portion comprising the double-stranded DNA and a single-stranded DNA molecule containing a nucleotide sequence substantially homologous to a specific region of the nucleotide sequence of the double-stranded DNA or a PNA containing a nucleotide sequence substantially homologous to a specific region of the nucleotide sequence of the double-stranded DNA and of cleaving any one of the phosphodiester bonds in or adjacent to the three-stranded DNA portion,
(b) a homologous recombination protein,
(c) nucleoside triphosphate or its analogue, and
(d) optionally a buffer.
Herein, nucleic acids, peptide nucleic acids (PNA), nucleotides, and other compounds or reagents are sometimes abbreviated following the conventional manner in the technical field.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.